Int.J.Curr.Microbiol.App.Sci (2015) 4(6): 201-210
ISSN: 2319-7706 Volume 4 Number 6 (2015) pp. 201-210
http://www.ijcmas.com
Original Research Article
Biomolecular Characterization of Different Fenugreek Genotypes
(Trigonellafoenum-graecum L.)
Jignesh Patel, J. J. Dhruve* and J.G.Talati
Department of Biochemistry, Anand Agriculture University,
Anand, Gujarat, India
*Corresponding author
ABSTRACT
Keywords
Trigonella
foenumgraecum L.,
Methi, Greek
hay, Antioxidant
activity,
secondary
metabolites
In the present investigation biomolecular characters in fenugreek seeds were
estimated. Results revealed minimum moisture content in genotype JFG.261.
Genotype PEB and JFG.261 recorded the higher ash and oil content, respectively.
The lowest and the highest phenol content were recorded in the genotype JFG.266
and kasturi methi, respectively. Significantly higher total antioxidant activity was
found in genotype kasturi methi. The maximum total soluble sugars and reducing
sugars were observed in Guj. methi 2. Genotype PEB estimated the highest free
amino acid content with regard to the protein content in the seeds. True protein and
crude protein content was recorded maximum in genotype JFG.260 and kasturi
methi respectively.
Introduction
hard yellow embryo surrounded by a
corneous and comparatively large layer of
white semi-transparent endosperm (Betty,
2008).
Fenugreek (Trigonellafoenum-graecumL.) is
a self-pollinating crop, which is the native
plant of the Indian subcontinent and the
eastern Mediterranean region. It is an old
medicinal plant and has been commercially
used as a traditional spice and medicine.
Fenugreek is originally from southern
Europe and western Asia, but today it is
grown in many parts of world (Naidu et al.,
2011).
India is the major producer and exporter of
fenugreek. This crop is mainly grown in arid
as well as semi-arid region of India. In India,
it is grown in 93090 ha area with production
of 112845 tonnes and productivity is 1.21
tonnes/ha (Balraj Singh, 2014). In India the
major fenugreek growing states are
Rajasthan, Gujarat, Madhya Pradesh,
Tamilnadu, Uttar Pradesh and Punjab. More
than 80% area and production is contributed
by Rajasthan state alone. The major districts
Fenugreek is known for its pleasantly bitter
seeds. The seeds available in whole ground
form are used to flavour in many foods
including curry powders, spice blends and
teas. The fenugreek seeds contain a central
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Int.J.Curr.Microbiol.App.Sci (2015) 4(6): 201-210
growing fenugreek in Gujarat state are Patan
and Dahod.
Materials and Methods
The present experiment was conducted at
the Biochemistry department, B. A. College
of
Agriculture,
Anand
Agricultural
University, Anand (Gujarat) The materials
for study consisted of
thirteen
genotypes/cultivars viz., JFG.179, JFG.226,
JFG.234, JFG.240, JFG250, JFG256,
JFG.260, JFG.261, JFG263, JFG.266,
GUJARATI-METHI.2, PEB, and KASTURI
METHI were obtained from Main Vegetable
Research Station, Anand Agricultural
University, Anand.
Fenugreek seeds were used as a spice in
food preparation as well as an ingredient in
traditional medicine. The seeds are vice
source of calcium, iron, ß-carotene and other
vitamins (Sharma and Raghuram, 1990).
Fenugreek seeds and leaves should be
included in normal diet of family, especially
diet of growing kids, pregnant ladies,
puberty regarding girls and elder members
of family because they have haematinic
value (Bukhari et al., 2008). Fenugreek seed
is widely used as galactagogue i.e. milk
producing agent by nursing mother to
increase inadequate breast supply. Many
scientist have recorded that the seeds also
contain lysine and L-tryptophan rich
proteins, mucilaginous fibres and other
biochemical such as saponin, coumarins,
sapogenins, phytic acids etc. which are
thought for many of its presumed
therapeutic effects, many inhibit cholesterol
absorption and thought to help lower sugar
levels. Therefore fenugreek seeds are used
as traditional remedy for the treatment of
diabetes and hyper cholesteroleria in Indian
medicines (Bhukri et al., 2008, Sauvaire et
al., 1991 and Basch et al., 2003).
The presence of flavonoids, cardiac
glycoside, steroids, terpenoids and tannins in
the fenugreek seed was determined by the
methods described by Sofowara (1993) and
Harborne (1973).The methods of A.O.A.C
(1984) were used to determine the Moisture
content and total ash. The oil content was
determining using Petroleum ether in
Soxhlet reflux extractor (Chaturvedi and
Sanker, 2006). For the estimation of phenols
the method as proposed by Malick and
Singh (1980). The total antioxidant activity
was determined by FRAP (Ferric reducing
antioxidant power) assay (Varga et al.,
1998). The true protein content in the seed
was estimated by Lowry et al. (1951) where
as crude protein (N x 6.25) was determined
using micro Kjeldahl method (A.A.A.C,
2000). The free amino acid was determined
using the method suggested by Moore and
Stein (1948). Reducing sugars were
estimated by the method of Nelson (1944).
Total soluble sugar was estimated by phenol
sulphuric acid method (Sadasivam and
Manickam, 1992). The content of nonreducing sugar was calculated from the
difference between soluble sugar and
reducing sugar.
The purpose of this study was to evaluate
fenugreek as new potential source for its
higher
nutraceutical
value.
The
mucilaginous seeds have medicinal values
as a tonic, emollient, carminative, diuretic,
restorative etc. (Duke, 2007). Now a days
search for natural nutraceutical source is
gaining much important. Thus it is important
to identify new sources of safe and
inexperience nutraceutical constituents of
natural
origin.
Hence,
in
present
investigation an attempt has been made to
identify fenugreek genotypes for its higher
nutraceutical value through bio molecular
characterization.
The varieties of seed proteins was analysed
by using SDS-PAGE. For extraction of
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Int.J.Curr.Microbiol.App.Sci (2015) 4(6): 201-210
protein each genotypes had three
replications and 25 seed for every
replication was randomly selected and
ground to fine powder with motor and
pestle, and a total of 150µl sample buffer
was added to a 0.05gm seed powder and
mixed thoroughly by vortex in eppendorf
tube (1.5ml) with a small glass rod. The
extraction buffer contained 0.05 M Tris base
6.5 g/L; 0.007 M citric acid (monohydrate)
1.5 g/L; 0.1% cysteine hydrochloride 1 g/L;
0.1% ascorbic acid (Na salt or free acid) 1
g/L; 1.0% polyethylene glycol (H 3500) l0.0
g/L; 1 mM 2- mercaptoethanol 0.08 mL/L,
the final pH 8.0 (Arulsekar and Parfitt,
1986), centrifuged at 18000rpm for 20min.
to monitor the movement of the protein in
gel, Bromophenol blue was used as a
tracking dye. Seed protein was analysed
through
SDS-PAGE
using
12%
polyacrylamide gel. The molecular weight
dissociated polypeptides were determined
using protein standard (M.W SDS- 2 to 150
kDa) of Merck, India. The gels were stained
with Coomassie brilliant blue and distained
till the background became transparent.
extracts. The phytochemical tests reveal the
presence of various bioactive secondary
metabolites which might be responsible for
their medicinal attributes. Quantitative
assessments of the different phytochemicals
detected during investigation was graded as
ve for 0, +ve for 1, ++ve for2 and +++ve
for 3. The observations and inferences made
in the phytochemical tests are presented as
follows:
Flavonoids: Flavonoids are acts as antiallergic, anti-inflammatory, anti-microbial
and anti-cancer activity. A yellow coloration
was observed in all the fenugreek genotypes
except JFG-261 indicating thereby the
presence of flavonoids in all fenugreek
genotypes screened.
Cardiac glycosides: Cardiac glycoside is
useful for heart failure and cardiac
arrhythmia (Denwick, 2002). A brown ring
obtained at the interface indicated the
presence of a de-oxy sugar characteristic of
cardenolide i.e. Cardiac glycosides was
observed in seven genotypes of fenugreek
out of thirteen genotypes.
Result and Discussion
Steroids: Steroids are important for cardiotonic activity. Steroid possesses insecticidal
and antimicrobial properties (Callow, 1936).
A reddish brown ring at the interface was
observed with the extract of all fenugreek
genotypes/cultivars except JFG-261 plants
indicating the presence of steroids.
Phytochemical screening
Phytochemicals means the chemicals which
may have biological importance but are not
established as important nutrients. In a
narrower sense the terms phytochemical
describe the number of secondary metabolic
compounds found in plants. Scientists
estimate that about 10,000 different
phytochemicals having the capability to
have an effect on diseases like cancer and
metabolic syndrome etc.
Terpenoids: Terpenoidshave been found to
be useful in the prevention and therapy of
several
diseases,
including
cancer.
Terpenoids are also known to possess
antimicrobial, antifungal, antiparasitic,
antiviral, anti-allergenic, antispasmodic,
anti-hyperglycaemic, anti-inflammatory and
immunomodulatory properties (Rabi et al.,
2009; Wagner and Elmadfa, 2003). A deep
The result of the preliminary phytochemical
screening from fenugreek seeds (Table 1)
shows
the
presences
of
different
phytochemicals prepared indifferent solvent
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Int.J.Curr.Microbiol.App.Sci (2015) 4(6): 201-210
red colour was observed in ten genotypes of
fenugreek out of thirteen genotypes.
The lowest ash content found in genotype
JFG.263 (1.25%) and the highest found in
genotype PEB (5.23%). The non-significant
differences were recorded among genotype
JFG.226, JFG.250, JFG.179 and JFG.266.
The presence of ash in fenugreek plant in
such quantities are satisfying, because of the
high importance of mineral for health
maintenance and development, where the
minerals are essential for human body, they
are basic content of many of body tissues,
such as calcium and phosphorous for bone
and iron for blood and muscles. Ash
consider basic element of biomolecules in
addition to their roles in connectivity
process and in all of biochemical reaction
(Mann Jim and Truswell Stewart, 2002).
Tannins: Plant tannins are a large, diverse
group of polyphenolic compounds found
throughout several species in the plant
kingdom. Tannins have a protective function
in the bark of the roots and stems, or any
outer layers of plants. They are astringent in
nature due to their high polyphenol content.
This attribute confers the ability to form
strong complexes with proteins, starches and
other macromolecules (Catherine Clinton,
2009). Tannins are able to inhibit HIV
replication selectivity and are used as
diuretic. Tannin has recognized for
pharmacological properties and make trees
and shrubs a difficult meal for many
caterpillars (Heslem, 1989). A green
precipitate was observed in only Kasturi
Methi indicating thereby the presence of
tannins in only this genotype analysed.
Oil
The data on fenugreek seed oil is presented
in Table 2. The oil content was varied from
2.56% to 4.74%. Significantly the highest
oil percentage was recorded in JFG.261
(4.74%) and lowest oil percentage was
recorded in Gujarat methi-2 (2.56%). The
genotype JFG.266, Kasturi methi, JFG.179,
JFG.263, JFG.266, PEB and JFG.240 were
recorded at par.
Moisture
The mean value for genotypes (Table 2)
showed significantly the highest and the
lowest moisture content was found in JFG226 and JFG.260 (5.92%), respectively. The
genotypes JFG.261, Kasturi methi and
JFG.250 were recorded at par, in case of
genotype JFG.234 and JFG.240also
recorded more or less same. Singh and his
colleagues have studied the contribution of
fenugreek (Trigonellafoenum-graecumL.)
seeds
towards
the
nutritional
characterization. They have reported that the
mean moisture per cent in seeds were
recorded to be 8.86% and it was ranged
from 7.57 to 11.51%.
Phenol
The total phenol of different fenugreek seeds
cultivars were duplicated in table 2. The
content was varied from 1.69 to 3.22%. The
higher percentage of total phenol was
recorded in kasturi methi (3.22%) and lower
was observed in JFG.266 (1.69%).The
results indicate that vegetables containing
high phenolic may provide a source of
dietary
anti-oxidants.
The
phenolic
compounds may contribute directly to the
antioxidant action; therefore, it is necessary
to investigate total phenolic content
(Bukhari et al., 2008).
Ash
The present investigation revealed that the
ash content was found to be significant
(p<0.05) among all genotypes of fenugreek.
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Int.J.Curr.Microbiol.App.Sci (2015) 4(6): 201-210
sugars was recorded in JFG.250 (7.94%) and
the lowest non-reducing sugars found in
genotype JFG.226 (2.12%).
Total antioxidant activity
Total antioxidant activity of Fenugreek
seeds was calculated and data are presented
in Table- 2. The content was varied from 8.5
to 13.85 mg/g. significantly the highest
percentage of total antioxidant activity was
recorded kasturi methi (13.85 mg/g) and the
lowest was observed in JFG.261 (8.5 mg/g).
The genotype JFG.256 and JFG.226
recorded were at par. The reducing power of
bioactive compounds is associated with
antioxidant activity. So it is necessary to
determine the reducing power of phenolic
constituents elucidate the relationship
between the antioxidant effects and there
reducing power (Yildirim et al., 2001;
Siddhuraju et al., 2002). Our results are
agreement with results of Bukhari et al.
(2008). They have reported that all extracts
of the fenugreek exhibit antioxidant activity.
These findings suggest that the fenugreek
extracts could act as potent source of
antioxidants.
The principle function of carbohydrates is to
serve as a major source of energy for the
body. Each gram of carbohydrate yields 4
Kcal of energy regardless of its source. In
Indian diets 60 to 80% of energy is derived
from carbohydrates. The findings are in
consonance with Gopalan et al., (1992);
Kochhar et al., (2006); Saibaba and
Raghuram, (1977) and Sumayya et al.
(2012). Reducing sugars and non-reducing
sugar of plants varied from 0.78 to 4.43,
1.03 to 8.0. Lower total, reducing and nonreducing sugar and almost similar starch
content of fenugreek seed has been reported
by E1-mandy and Sebaiy (1983).
Free amino acid
The free amino acid in different genotype of
fenugreek seeds were depicted in Table- 3.
Significantly the highest and lowest
percentage of free amino acid was recorded
in PEB (13.85%) and JFG.240 (10.63%);
respectively. Sauvaire et al. (1984) have
observed that 4-hydroxy isoleucine represent
up to 80% of free amino acid in fenugreek
seeds. The concentration does not decrease
in later stage of maturation of the seeds, but
it absent from the seed reserve protein.
Total soluble sugars, reducing sugars and
non-reducing sugars
Total soluble sugars, reducing sugars and
non-reducing sugars of different fenugreek
seeds cultivars are depicted in Table- 3. The
content of total soluble sugars was varied
from 4.15% to 11.01%. Significantly the
highest percentage of total soluble sugar was
recorded in Gujarat methi-2 (11.01%) and
the lowest were recorded in JFG.226
(4.15%). The content of reducing sugars was
varied from 1.99% to 3.47%. Significantly
the highest percentage of reducing sugars
was recorded in Gujarat methi-2 (3.47%)
and the lower reducing sugars found in
genotype JFG.260 (1.99%) which was at par
with genotype JFG.226 and Kasturi methi.
The content of non-reducing sugars was
varied from 2.12% to 7.94%. Significantly
the highest percentage of non-reducing
True protein
The true protein in different genotype of
fenugreek seeds were recorded in Table- 3.
The content was varied from 7.26% to
11.26%. Significantly the highest and lowest
percentage of true protein was recorded in
JFG.260 (11.26%) and JFG.263 (7.26%);
respectively. The non-significant differences
were recorded among JFG.250, JFG.179,
JFG.234 and JFG.261.
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Int.J.Curr.Microbiol.App.Sci (2015) 4(6): 201-210
Table.1 Phytochemical screening of 13 different genotypes
Name of Genotype
Flavonoid
JFG.179
JFG.226
JFG.234
JFG.240
JFG.250
JFG.256
JFG.260
JFG.261
JFG.263
JFG.266
Guj.methi 2
PEB
KM
1
1
1
1
3
1
1
0
1
3
1
2
2
Cardiac
glycoside
0
0
0
1
3
1
0
0
2
3
0
2
3
Steroid
Terpenoid
Tanin
3
2
1
1
1
1
2
0
1
1
2
1
3
1
1
0
0
3
1
3
0
2
3
2
3
2
0
0
0
0
0
0
0
0
0
0
0
0
1
(3) High presence; (2) Moderate; (1) Low; (0) absent
Table.2 The moisture, ash, oil, phenol and total antioxidant activity in different fenugreek seeds
Genotype
Moisture (%)
Ash (%)
Oil (%)
Phenol (%)
JFG.179
JFG.226
JFG.234
JFG.240
JFG.250
JFG.256
JFG.260
JFG.261
JFG.263
JFG.266
Guj.methi-2
PEB
Kasturi methi
S.Em±
C.D. at 5%
CV%
9.58
13.63
10.75
10.57
7.8
8.74
5.92
7.18
6.86
8.71
8.33
6.73
7.47
0.129
0.376
2.592
2.22
2.16
2.56
3.30
2.20
2.51
2.69
2.90
1.25
2.23
5.05
5.22
3.80
0.109
0.318
6.456
3.16
2.56
3.62
3.43
4.47
3.59
4.26
4.74
3.25
3.34
2.56
3.35
3.05
0.317
0.922
15.715
2.56
1.98
2.38
2.52
2.07
2.36
1.90
1.98
1.78
1.69
1.71
1.72
3.22
0.047
0.137
3.804
206
Total
Antioxidant
Activity(mg/g)
11
9.25
10.5
10.9
10
9.21
10.62
8.5
9.71
9.9
11.7
11.5
13.8
0.034
0.100
0.566
Int.J.Curr.Microbiol.App.Sci (2015) 4(6): 201-210
Table.3 Total soluble sugar, reducing sugar, total protein, crude protein, free amino acid in
different fenugreek seeds
Genotype
Total Soluble
Sugar (%)
JFG.179
JFG.226
JFG.234
JFG.240
JFG.250
JFG.256
JFG.260
JFG.261
JFG.263
JFG.266
Guj.methi-2
PEB
Kasturi methi
S.Em±
C.D. at 5%
CV%
6.33
4.15
5.87
8.36
10.22
7.40
7.45
8.66
9.12
8.82
11.01
7.90
9.26
0.01
0.28
2.1
Non
Reducing Sugar
reducing
(%)
sugar (%)
1.99
2.03
2.51
2.36
2.28
2.40
2.51
2.49
2.54
2.29
3.47
3.07
2.04
0.02
0.07
1.62
4.34
2.12
3.35
5.99
7.94
5.00
4.98
6.17
6.66
6.53
7.54
4.82
7.22
0.154
0.78
2.20
Free Amino
Acid (%)
True Protein
(%)
Crude
Protein (%)
12.04
11.58
12.18
10.63
11.93
13.37
12.55
12.20
12.58
12.35
11.02
13.85
11.16
0.05
0.15
0.72
9.62
10.17
10.01
8.63
9.14
8.56
11.26
9.86
7.26
10.91
10.45
8.02
9.17
0.03
0.1
0.57
14.23
14.73
17.50
18.67
21.56
17.65
23.34
22.90
21.15
22.47
27.57
26.40
32.24
0.25
0.73
2.00
Fig.1 Dandogram of 13 different fenugreek seeds genotype based on Jaccard s similarity
coefficients
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Int.J.Curr.Microbiol.App.Sci (2015) 4(6): 201-210
Plate.1 SDS PAGE banding pattern of different genotype of fenugreek seeds
1
3
1
Srinivasan (2006) reported that cooking
does not affect the quality of fenugreek seed
proteins. It is evidenced in the animal study
that the replacement of casein diet up to
10% by fenugreek seeds did not produce any
harmful effect in protein quality of casein as
studies on animal subjects has been
evidenced for protein efficiency ratio,
protein digestibility and net protein
utilization, debitterized fenugreek seeds are
rich in protein and lysine.
Protein electrophoresis
The soluble protein of fenugreek seeds were
separated by sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDSPAGE) method. The banding pattern was
present in plate-1. The band no.1 was
present in genotype JFG.250 and JFG.256
with Rm value 0.130cm. In all genotypes
total eighteen bands having Rm value
ranged from 0.130 to 0.937 were recorded.
The band no.2 was present in all genotypes
except genotype JFG.263 and Kasturi methi.
The band no.3 having Rm value 0.247 was
present only in genotype Kasturi methi. In
case of band no.4, 5 and 6 was absent in
genotype Kasturi methi. Band no.9, 13, 15
and 18 were absent in genotype PEB. The
protein band no.7, 11 and 17 were present in
all fenugreek genotypes. Sanjeevrajan et al.
(2012) evaluated protein from various
legumes seeds using different protein
extraction methods. They have compared
seven different protein extraction methods
from the seeds of four different leguminous
plants.
Crude protein
The percentage of crude protein present in
different genotype of fenugreek seeds were
recorded in Table- 3.The content was varied
from 14.23% to 32.24%. Significantly the
highest percentage of total protein was
recorded in kasturi methi (32.24%) and the
lower percentage of crude protein was
recorded in JFG.179 (14.23%), which was at
par with JFG.226. The genotype JFG.263
and JFG.250 recorded were at par. The
estimation of nitrogen was done by Kjeldahl
method wherein the protein content was
obtained by multiplying the nitrogen value
with 6.25, (A.O.A.C., 1990). Gopalan et al.
(1992) reported that fenugreek seeds richest
source of crude protein (25.8%).
Jaccard s similarity coefficient on the basis
of presence and absence of bands was
calculated for all possible pairs of 13
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Int.J.Curr.Microbiol.App.Sci (2015) 4(6): 201-210
genotypes of fenugreek. The highest
similarity Index value 1.0 was found among
genotype JFG.179, JFG.226 and JFG.250.
The highest similarity index was also found
between genotype JFG.240 and JFG.263,
while the minimum similarity index value
0.40 was found between JFG.266 and
guj.methi-2.
Basch, E., Ulbricht, C., Kuo, G., Szapary, P.,
Smith,
M.
2003.
Therapeutic
applications of fenugreek. Altern. Med.
Rev., 8: 20 27.
Betty, R.I. 2008. The many healing virtues
of fenugreek. Spice India, 1: 17 19.
Bukhari, S.B., Bhanger, M.I., Memon, S.
2008. Antioxidative activity of extracts
from
fenugreek
seeds
(Trigonellafoenum graecum). Pak. J.
Anal. Environ. Chem., 9: 78 83.
Callow, R.K. 1936. Steroids. Proceedings of
the Royal Society of London Series
A 157 194.
Catherine Clinton, N.D. 2009. Plant tannins:
A novel approach to the treatment of
ulcerative colitis. Nat. Med. J., 1(3): 1
4.
Chaturvedi, R.K., Sankar, K. 2006.
Laboratory
Manual
for
the
physicochemical analysis of soil, water
and plant. Wild life institute of India,
Dehradun L. 64 Pp.
Denwick, P.M. 2002. Natural products: A
biosynthetic approach. 2nd edn. John
wiley and sons, Ltd, England. Pp. 241
243.
Duke, A. James, Duke Peggy Ann, K., Du
Cellie Judith, L. 2007. Duke s
handbook of medicinal plant of the
Bible. CRC press Taylor and Francis
Group, printed in the united states of
America.
El-Mahdy, A.R., El-Sebaiy, L.A. 1983.
Changes
in
carbohydrates
of
germinating
fenugreek
seeds
(Trigonellafoenumgraecum L.). J. Sci.
Food Agricult., 34: 951 956.
Gopalan,
C.,
Rama
Sastri,
B.V.,
Balasubramanian, S.C. 1992. Revised
and updated by Narasingha Rao B.S.,
Deosthale, J.G. and Pant, K.C.
Nutritive value of Indian foods. NIN,
ICMR, Hyderabad, India.
Over all it can be concluded that from
present experiment that seeds of kasturi
methi contain higher amount of cardiac
glycoside and steroid whereas moderate
amount of flavonoids and terpenoids.
Flavonoids and steroids were present in all
genotypes whereas tannin was only present
in kasturi methi. The genotypes JFG.250 and
JFG.266 contain higher amount of
flavonoids,
cardiac
glycoside
and
terpenoids. JFG.261 genotype doesn t
contain any of phytochemicals. Kasturi
methi registered with higher relative water
content, phenol, crude protein and Total
antioxidant activity. Seeds of PEB have
maximum ash and free amino acids. Thus
this genotype may be used for breeding
purpose.
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